Novarro was born José Ramón Gil Samaniego on February 6, 1899 in Durango City, Durango, Mexico, to Dr. Mariano N. Samaniego, and his wife, Leonor (Gavilan). The family moved to Los Angeles, California, to escape the Mexican Revolution in 1913. Novarro's direct ancestors came from the Castilian town of Burgos from where two brothers emigrated to the New World in the seventeenth century.

He entered films in 1917 in bit parts. He supplemented his income by working as a singing waiter. His friends, actor and Director Rex Ingram and his wife, the Actress Alice Terry, began to promote him as a rival to Rudolph Valentino, and Ingram suggested he change his name to "Novarro." From 1923, he began to play more prominent roles. His role in Scaramouche (1923) brought him his first major success.

Novarro was troubled all his life by his conflicted feelings toward his Roman Catholic religion and his homosexuality. His life-long struggle with alcoholism is often traced to these problems. In the early 1920s, Novarro had a romantic relationship with Composer Harry Partch, who was working as an usher at the Los Angeles Philharmonic at the time, but Novarro broke off the affair as his acting career began to become successful. MGM mogul Louis B. Mayer reportedly tried to coerce Novarro into a "lavender marriage", which he refused. He was romantically involved with Journalist Herbert Howe, who was also his publicist in the late 1920s, and with a wealthy man from San Francisco, Noël Sullivan.

In 1925, Novarro achieved his greatest success in Ben-Hur. His revealing costumes caused a sensation. He was elevated into the Hollywood elite. As did many stars, Novarro engaged Sylvia of Hollywood as a therapist (although in her tell-all book, Sylvia erroneously claimed that Novarro slept in a coffin). With Valentino's death in 1926, Novarro became the screen's leading Latin actor, though ranked behind his MGM contemporary, John Gilbert, as a leading man. He was popular as a swashbuckler in action roles and considered one of the great romantic lead actors of his day. Novarro appeared with Norma Shearer in The Student Prince in Old Heidelberg (1927) and with Joan Crawford in Across to Singapore (1928).

He made his first talking film, starring as a singing French soldier, in Devil-May-Care (1929). He starred with Dorothy Janis in The Pagan (1929), with Greta Garbo in Mata Hari (1931), with Myrna Loy in The Barbarian (1933) and opposite Lupe Vélez in Laughing Boy (1934).

When his contract with MGM Studios expired in 1935 and the studio did not renew it, Novarro continued to act sporadically, appearing in films for Republic Pictures, a Mexican religious drama, and a French comedy. In the 1940s, he had several small roles in American films, including We Were Strangers (1949), directed by John Huston and starring Jennifer Jones and John Garfield. In 1958, he was considered for a role in the television series The Green Peacock, with Howard Duff and Ida Lupino, after their CBS Television sitcom Mr. Adams and Eve (1957–58). The project, however, never materialized. A Broadway tryout was aborted in the 1960s. Novarro kept busy on television, appearing in NBC's The High Chaparral as late as 1968.

Novarro was murdered on October 30, 1968, by brothers Paul and Tom Ferguson, aged 22 and 17, who called him and offered their sexual services. He had in the past hired prostitutes from an agency to come to his Laurel Canyon home for sex, and the Fergusons obtained Novarro's telephone number from a previous guest.

Prize-winning Greek Playwright Pavlos Matesis wrote a play in two parts titled "The Ghost of Mr. Ramon Novarro", which was first staged at the National Theatre of Greece in 1973.

In late 2005, the Wings Theatre in New York City staged the world premiere of Through a Naked Lens by George Barthel. The play combined fact and fiction to depict Ramon Novarro's rise to fame and his relationship with Hollywood Journalist Herbert Howe.

According to the prosecution in the murder case, the two young men believed that a large sum of money was hidden in Novarro's house. The prosecution accused the brothers of torturing Novarro for several hours to force him to reveal where the non-existent money was hidden. They left the house with $20 they took from his bathrobe pocket. Novarro died as a result of asphyxiation—having choked to death on his own blood after being beaten. The two perpetrators were caught and sentenced to long prison terms, but released on probation in the mid-1970s. Both were later re-arrested for unrelated crimes for which they served longer prison terms than for the murder of Novarro. Scotty Bowers' 2012 autobiography Full Service: My Adventures in Hollywood and the Secret Sex Lives of the Stars provides questionable details about Novarro's death. In a 1998 interview, Paul Ferguson finally assumed the blame for Novarro’s death.